(a) Field of the Invention
This invention relates to microspheres, and to the method of producing the same. More particularly the present invention is concerned with the production of an immobilizing material, such as calcium alginate beads.
(b) Description of Prior art
The immobilization or isolation of components in small spherical particles has proven to be of great utility in a wide variety of industrial applications. Ever since the development by National Cash Register Corp. of microencapsulated inks for carbonless copies in the 1950's [1], many techniques have been developed for immobilizing other hydrophobic solvents, high molecular weight water soluble molecules (proteins, polymers, hemoglobin) as well as living cells [2].
Many early immobilization methods involved the use of organic solvents or other chemicals that were incompatible with many potential biological encapsulants [3]. The use of various gel forming proteins (collagen and gelatin) and polysaccharides (agar, calcium alginate, and carrageenan) introduced a milder, biocompatable immobilization system [4]. This method involved heating the gel until liquefaction (40.degree.-60.degree. C.) occurred, adding the material to be immobilized, and cooling the solution until it solidified. However, this procedure has several drawbacks since the high temperatures used could prove to be incompatible with thermally labile material as well as the resulting gel must be cut into small pieces.
A more gentle and practical system has been developed that involves adding an ionic polysaccharide solution containing the material to be immobilized dropwise through a syringe needle into a solution of a divalent cation (typically CaCl.sub.2), the ion crosslinking the charged species on the polysaccharide and thus forming an insoluble gel bead [5]. This system typically uses alginate as the ionic polysaccharide and has been popular for immobilizing many diverse materials such as plant [6] and mammalian [7] cells, yeast [8], bacteria [9], insulin [10], toners [11], magnetite [12] as well as producing food products like artificial caviar [13].
This method will produce microspheres of uniform size. However, it has three drawbacks; the first being that reduction in microsphere diameter is limited by the syringe needle diameter as well as the viscosity of the solution, with microspheres less than 1.0 mm being difficult to produce. Smaller microspheres have the advantage that small molecules can diffuse in or out of the beads at higher rates (less mass transfer limitations). Their use would therefore result in more rapid reaction rates and microsphere rupture resulting from possible gas production (such as CO.sub.2 in fermentation) would be eliminated. Also, if the microspheres were used for controlled release of a particular substance, this release would occur more rapidly.
The second drawback of the drop technique is that the microspheres tend to be teardrop-shaped due to drag forces on the alginate droplet when it solidifies following impact with the solidifying solution (CaCl.sub.2).
Finally, the third limitation of this drop technique is that it is not suitable for industrial scale-up. To manufacture microspheres on a large scale, a large number of needles would have to be operated concurrently.
Several techniques to alleviate the first problem have been developed. Air jets impinging on the needle, electrostatic systems, and rotating or vibrating needles have been examined and do produce smaller microspheres (down to &lt;0.5 mm). Atomizing spray techniques have also been developed. This latter technique does produce smaller microspheres at higher rates but shearing effects in such a system could prove to be harmful to many biological species.
Producing microspheres via emulsion techniques have been utilized in other production methods but have only been used on a few occasions with ionic polysaccharides. One technique involves a hot carrageenan/oil emulsion that is dropped into cold water [14]. Another method involves cooling this emulsion in an ice bath [15]. An oil-in-aqueous alginic acid emulsion can also be added dropwise to a CaCl.sub.2 solution to encapsulate oil droplets in alginate [16]. The former techniques have the heat requirement disadvantage whereas the latter still requires a drop technique.
The difficulty of using emulsion techniques with ionic polysaccharide/CaCl.sub.2 is that both reactants are insoluble in the oil phase. An emulsion of a polysaccharide aqueous solution in oil can be added to a CaCl.sub.2 solution [15]. This technique will produce capsules, however, diameters cannot be controlled and the capsules tend to coagulate into large masses before hardening properly.